Insect-borne diseases are a huge and growing problem in world health. The most serious of these is malaria, which affects over 300 million people each year, and kills over one million people. Although the mortality occurs among young children in sub-Saharan Africa, malaria still poses a health risk in the United States, with approximately 3000 cases being reported in 2000-2001 alone.
An important part of a multifaceted approach to this problem is control of the insects that act as vectors for the transmission of disease. A primary means of limiting insect vectors is limiting the number of insects in the population through the use of insecticides. There are two large concerns with regard to the use of insecticides. One is the ability of insects to evolve a resistance to insecticides, which happens frequently. The other concern relates to the general safety and efficacy of the use of insecticides, specifically, some insecticides such as the group of insecticide compounds known as the organophosphates, affect vertebrate nervous systems, and therefore pose a risk to human and animal populations. Therefore, tools that help in the discovery of insecticides that have a broad-spectrum effect on insects across species and yet remain harmless to humans and other animals are very useful in this technical field.
An excellent model for the testing of genetic insecticide targets is Drosophila melanogaster. The Drosophila genome has been entirely sequenced and it is one of, if not the, most well studied and documented insects in the world. A wide array of genetic and molecular research methods and tools are available for efficacious use with Drosophila. In addition, many Drosophila genes and proteins are highly conserved across insect species, meaning that research on Drosophila genes and proteins will likely have an impact on other insect species as well.
When the Drosophila genome was surveyed for dosage sensitive regions only one was found which was both triplo-lethal and haplo-lethal. This locus, located in cytological region 83D4,5-E1,2, was called the Triplo-lethal locus, abbreviated Tpl. Stocks carrying a duplication of Tpl on one homolog and a deficiency on the other homolog are viable, and provide a powerful selection for either up or down mutations when crossed to a wild type fly. Using this selection, Keppy and Denell were able to obtain duplications and deficiencies of Tpl, but were unable to isolate point mutations following EMS or formaldehyde mutagenesis. Roehrdanz and Lucchesi were also unable to isolate point mutations of Tpl following EMS mutagenesis, although they did isolate mutations in the Suppressor of Tpl locus, which has been shown to encode the Ell protein, a general transcription elongation factor.
Three hypotheses have been proposed to explain the lack of point mutations at Tpl: 1) the locus is very small so the mutation rate is very low, 2) the locus does not encode a protein so it is less sensitive to single base changes, and 3) the locus consists of a cluster of genes with at least partial redundancy, such that mutating one of the genes does not rescue the lethality caused by a duplication of the entire cluster. The small size hypothesis predicts that as the number of mutagenized chromosomes increases, eventually mutants will be found. However, we have subsequently screened more than 106 chromosomes and still have not isolated point mutants. The non-protein-coding hypothesis predicts that transposon insertions would inactivate the locus, as would inversions with a breakpoint in the gene, and in spite of considerable effort we have never isolated a P element insertional mutation in Tpl. Thus the most likely hypothesis is that Tpl consists of a cluster of genes with at least partial redundancy.
The complete genomic sequence of D. melanogaster has allowed us to test the prediction that the Triplo-lethal region contains a cluster of genes with high similarity. To do that, first we defined the molecular limits of Tpl by isolating and mapping duplications and deletions and then examining the sequence within those limits for repeated genetic units. We describe here the discovery of a multi-gene family in the Triplo-lethal region, consistent with the best hypothesis based on the genetic data. The proteins encoded by this family are novel, although the protein sequences have features that allow us to make predictions about their function. We would expect that a family of genes whose dosage is so critical would be well conserved, and would show evidence for strong selection on expression levels. Comparison of the Drosophila melanogaster gene family with the orthologous genes in Anopheles gambiae allows us to analyse the expression, selection and evolution of the family.